The overall objective to this proposal is to characterize, in a differentiated mammalian cell type, a newly recognized biochemical pathway for regulation of intracellular Ca2+ and to establish the functional significance of this signalling system in endocrine secretory cells. In the sea urchin egg, it has been established that a novel metabolite of NAD+, called cyclic ADP-ribose (cADPR), releases intracellular Ca2+ from a non-Ins (1,4,5)P3-sensitive pool with a potency that is comparable to that of IP3. We have demonstrated that a cADPR-like molecule is synthesized by several rat tissues including the pituitary gland. We have also shown, for the first time, that authentic cADPR releases sequestered Ca2+ by a non IP3 dependent mechanism in permeabilized rat pituitary cells of the GH4C1 strain. Based on these findings, we propose to test the hypothesis that cADPR is a functional mediator of cytosolic Ca2+ regulation in mammalian pituitary cells. There are 4 Specific Aims. 1. To prove that authentic cADPR is synthesized by intact pituitary cells. 2. To investigate the control of cellular cADPR concentrations in response to secretagogue agonist hormones. 3. To establish the functional role of cADPR in regulation of [Ca2+}i and hormone secretion. And 4. To determine the biochemical mechanism of action of cADPR. The results of these experiments will contribute fundamental new knowledge on the complex regulation of intracellular Ca2+ in mammalian cells which is not fully explained by the inositol lipid pathway or by alterations in plasma membrane Ca2+ fluxes. Such understanding is important because [Ca2+]i plays such a pivotal role in cellular secretion, DNA synthesis, cell division, motility, expression of specific genes (e.g., prolactin), and regulation of a number of critical intracellular enzymes. Understanding further this new limb of intracellular Ca2+ regulation should prove insightful in designing new pharmacological approaches to modulation of specific cell functions.